A novel phosphorylated spin trap, 5-diethoxyphosphoryl-5-methyl 1-pyrroline N-oxide (DEPMPO), was used to monitor superoxide formation from purified endothelial nitric oxide synthase (eNOS). eNOS consists of a flavin-containing reductase domain and a heme-containing oxidase domain that binds L-arginine. eNOS-dependent superoxide formation can be stimulated by redox cycling agents such as lucigenin or paraquat. The reductase domain is responsible for superoxide formed from these compounds. The superoxide adduct of DEPMPO is 15 times more stable than that of the more commonly used spin trap, DMPO. Moreover, unlike the DMPO-superoxide adduct, the DEPMPO-superoxide adduct does not spontaneously decay to the DEPMPO-hydroxyl adduct. Consequently, the steady-state level of the DEPMPO-superoxide adduct is higher than the DMPO-superoxide adduct at the same rate of superoxide generation. Thus, ESR spin trapping with DEPMPO using a loop-gap resonator is a viable approach to detect superoxide formation from eNOS-dependent bioactivation of xenobiotics.